Programmed cell death occurs in many tissues of our bodies throughout life. The quick and efficient removal of dying cells is a critical step that protects neighboring cells, and is also an important part of tissue remodeling and wound healing. The failure to remove apoptotic bodies has been implicated as a cause for certain types of chronic inflammation and autoimmune disorders. In mammals, macrophages and dendritic cells, as well as many other cells types can carry out the engulfment of apoptotic corpses. While several surface receptors that participate in the recognition of apoptotic cells have been identified, relatively little is known about the intracellular signaling events that regulate the uptake process itself and its downstream consequences. Recent cloning of the engulfment genes in the model organism C. elegans, and the identification of their homologues in mammals, has provided an exciting opportunity to delineate this process in better detail. The major focus of this proposal will be to determine the signaling events mediated through one of the evolutionarily conserved engulfment proteins GULP and to gain a better molecular understanding of the process of engulfment of apoptotic cells in mammals. CED-6 was identified as a candidate cytoplasmic adapter protein involved in engulfment of apoptotic cells in C. elegans, ced-6 is in the same functional genetic pathway that includes two other genes, ced-1 and ced-7, both of which encode membrane proteins. Elegant worm genetic studies have shown that ced-6 expression is required in the engulfing cell and not in the apoptotic cell and that ced-6 functions downstream of ced-1. Our laboratory has cloned the murine homologue of ced-6, denoted gulp, and has performed initial characterization of GULP protein in mammalian engulfment. In Aim 1, we will use the J774 macrophages and primary macrophages to perform structure: function studies of GULP and understand the role of GULP in anti-inflammatory cytokine production following engulfment. Several lines of evidence suggested CD91/LRP 1 as a GULP interacting protein, and LRP likely represents the CED- 1 homologue in mammals. The focus of Aim2 will be to determine the biological significance of GULP: LRP interaction through the use of LRP deficient cells lines in vitro, and conditionally targeted LRP deficient mice in vivo. To understand GULP function at the whole animal level, in Aim3, we will generate GULP knockout mice, as well as knock-in mice in which the LZ region is specifically mutated. In these mouse studies, we will determine the effect on engulfment due to the absence of GULP, or disrupted GULP function, and any potential effects on autoimmunity due to defective engulfment.